The high costs of petroleum, along with the increasing harmful environmental effects due to its use, have led researchers to search for renewable sources of energy and fuels. Up to now, established renewable energy sources, such as wind turbines and solar photovoltaics, focus on the production of electrical energy. Since as much as 30% of all energy consumption is in the form of liquid transportation fuels, a need exists to produce liquid fuels in a renewable way (US Energy Information Administration, 2011).
The chemical complexity of liquid fuels (e.g., ethanol or higher organic alcohols and alkanes) demands using sets of complex catalysis reactions that are best achieved through microbial enzymatic processes. The precursors of these liquid fuels could be simple gases, including hydrogen (H2), methane (CH4), or syngas (CO/H2/CO2), or any combination of the above gases (including CO/H2 or CO2/H2). Among the gases, H2 could become an important precursor to liquid fuels, because it can be produced from water electrolysis, for which the electrical power can be derived from renewable sources or from biomass, which is inherently renewable. CH4 is of especially high interest because it has become readily available from hydraulic fracturing and can be produced by fermentation of biomass.
Acetogens comprise a unique group of anaerobic bacteria that utilize the Wood-Ljungdahl pathway to convert H2/CO2, syngas (H2/CO) and a variety of sugars to produce predominantly acetate, along with the versatility to produce ethanol, butanol and other higher acids and alcohols through fermentative pathways (Drake, 1994). Acetogens have been employed for production of valuable chemicals from gaseous substrates at a commercial scale (Munasinghe and Khanal, 2010; Latif et al, 2014). Table 1 provides a selective list of acetogens able to utilize syngas, along with their titer values of the respective chemical end product, including current industrial potential.
TABLE 1Microbiological capabilities of various mesophilic pure strains of acetogenicbacteria using syngas (H2 + CO2/H2 + CO) to produce value added chemicals.PrimarymetabolicConc.Temp.pHOrganismproducts(g L−1)(° C.)profileObservationsAcetobacteriumAcetate44306.2Not capable ofwoodiigenerating otherproductsClostridiumEthanol,48375.8Maximum ethanolljungdahliiacetate,(ethanol)capacity 100 gallons perbutyratedry ton of waste biomassClostridiumEthanol,25.2632-376.1Severe limitation forragsdaleiacetate(ethanol)ethanol production belowpH 6ClostridiumEthanol,28375.2Gas transfer incoskatiiacetate(ethanol)suspension reactors12.5limited due to lower KLa(acetate)of gas transferClostridiumEthanol0.26-0.32376.4Lack of specificity withautoethanogenum2,3 butanediol andbutanol produced in largequantitiesAcetobacteriumEthanol 1.7377.7-8Only alkaliphilic strainbacchibut with limited capacityMoorellaAcetate 7.1606.4Limited capability tothermoaceticaproduce significantacetate from H2/COThermoanaero-Ethanol,21 mol606.63:1 stoichiometry forbacter kivuiacetateethanol/molCO:ethanolacetateAcetogens that are capable of using H2/CO2 or syngas belong to two categories: mesophilic (32-37° C.) and thermophilic (55-60° C.). While most studies have focused on unearthing the capabilities of mesophilic acetogens, several thermophilic acetogens, such as Thermoanaerobacter kivui and Moorella thermoautotrophica, have promise due to their high metabolic rates and capabilities.
Several challenges, however, limit the commercial application of acetogenic bacteria. For instance, higher gas-liquid mass transfer rates are needed. Gas-based metabolism by acetogens involves the utilization of relatively insoluble H2 and CO/CO2 for product synthesis, and a significant quantity of the gas is required to satisfy stoichiometric requirements. Stoichiometry indicates that the reactant gas requirements depend upon the nature of the specific microbial reaction, as indicated below for acetate and ethanol.
Acetate Production:4CO+2H2O→4CH3COOH+2CO2  (1)2CO2+4H2→4CH3COOH+2H2O  (2)
Ethanol Production:6CO+3H2O→4C2H5OH+4CO2  (3)2CO2+6H2→4C2H5OH+3H2O  (4)High-efficiency gas transfer to the acetogens is essential, since H2 and CO are very low-solubility gases. However, conventional designs to provide the highest volumetric gas transfer rates are not necessarily effective due to the significant energy consumption.
In addition to higher-efficiency gas transfer, achieving higher catalyst (active biomass) concentrations is also needed. Though it is preferable to have most of the carbon go to products rather than biomass, a certain cell concentration is needed to sustain industrially relevant production rates. Conventional suspended-cell recycling strategies using semi-permeable membranes have been plagued by fouling problems (Quereshi et al., 2005).
Methanotrophs represent another class of microorganisms that oxidize methane predominantly to CO2. However, under certain conditions such as nutrient limitation, these microorganisms store excess carbon and electrons in storage products such as polyhydroxybutytrate (PHB), which is a precursor to commercial plastics. These methanotrophs could also be metabolically engineered to produce alcohols and fatty acids too.
Process challenges when employing methanotrophs have been identified by the inventors and relate to the nature of the feed gases and delivery of such gases to a biofilm reactor. When employing methanotrophs, the mixture of the two gases required for their metabolism is CH4 and O2. Both gases are relatively insoluble in water (40 mg O2/L and 23 mg CH4/L at STP), making it difficult to dissolve either at high rates. In addition, practical biofuel applications will most likely obtain O2 from air, which has 20% O2 by volume, decreasing the solubility by a factor of 5. The use of air also brings an inert gas into the mixture, N2, which dilutes the reactants.
Another complication of using CH4 and O2 together is the flammability of this gas mixture. CH4 has a lower flammability limit (LFL) of 5% and a higher flammability limit (HFL) of 15% in air. If mixing these gases before delivering them to methanotrophs, the mixture should be either below the LFL or above the HFL to avoid the risk of combustion. Based on the stoichiometry of the reaction carried out by methanotrophs (shown below), an optimal mixture in air would be ˜9% CH4 balanced by air (˜18% O2), a flammable mixture.CH4+2O2+2H2O→CO2+4H2O  (5)
In order to achieve a non-flammable mixture, one approach would be to operate below the LFL of 5% CH4. This approach, however, introduces several serious drawbacks. First, the low concentration of CH4 in the gas significantly decreases its flux across the hollow fiber, which slows the overall reaction rate. Second, the addition of air creates a requirement for gas exhaust (mostly N2) from the system. The CH4 concentration of this exhaust gas would ideally be almost zero to minimize CH4 emissions and maximize CH4 utilization. However, this also implies that at least part of the reactor will encounter a very low CH4 concentration.
The challenges outlined above not only apply to acetogens and methanotrophs, but to any microbial metabolism that is based on feeding a gaseous substrate. New reactor configurations addressing these challenges should be considered for commercializing microbially driven gas fermentations/biotransformations. Other microorganisms able to use gaseous substrates aside from acetogens should also be considered.